Detection of alcaline isophosphatases by electrophoresis

ABSTRACT

The invention concerns a process for separating alkaline phosphatase (ALP) isoenzymes from a biological sample by electrophoresis, characterized in that the electrophoresis is carried out on an electrophoresis support after depositing a solution of lectin onto the electrophoresis support in a predetermined localised zone, under conditions which permit interaction between said lectin and the ALP isoenzymes contained in the analysed biological sample, deposition of the lectin solution further being carried out under conditions which are suitable to allow separation of the ALP isoenzymes constituted by the osseous fraction and by the hepatic fraction.

BACKGROUND OF THE INVENTION

[0001] Alkaline phosphatase (EC 3.1.3.1.) (abbreviation: ALP) is ametalloenzyme consisting of a group of isoenzymes present in differenttissues of animal organisms and in particular in man.

[0002] Alkaline phosphatase isoenzymes are important in protocols fordiagnosing different conditions in adults or in children, and a numberof methods have been proposed for separating and assaying suchisoenzymes. ALP isoenzymes can be divided into four classes:non-specific tissue (bone, liver and kidney), adult intestinal, foetalintestinal, and placental. A number of variations can exist within asingle class, namely:

[0003] hepatic: hepatic 1 (H1), hepatic 2 (H2);

[0004] ultrafast (UF);

[0005] osseous (Os);

[0006] placental: placental 1 (P1), placental 2 (P2);

[0007] intestinal: intestinal 1 (I1), intestinal 2 (I2), intestinal 3(I3).

[0008] Thus nine principal fractions can be distinguished which have tobe separated, identified and quantified in particular for theirdetection into hepatic and biliary disorders and into certain bonediseases, including osseous tumours or Paget's disease.

[0009] Reference will occasionally be made below to the term “fraction”to designate a class of ALP isoenzymes or a particular variant within aclass of isoenzymes. On the electrophoresis support, a “fraction”corresponds to a band revealed after migration.

[0010] The most frequent routine analysis carried out on alkalinephosphatase isoenzymes consists of measuring the total enzymaticactivity using a substrate of this enzyme, generallypara-nitrophenylphosphate. That method, however, cannot determine thelevels of the different isoenzymes.

[0011] The principal method for separative analysis of such compoundsuses electrophoretic techniques. Isoelectrofocussing is occasionallyused and can separate 10 to 20 bands depending on the procedure used.Identifying all of the bands is difficult, rendering clinicalinterpretation extremely awkward.

[0012] Zone electrophoresis enables a good separation of the principalforms of the isophosphatases. However, certain fractions aresuperimposed, in particular the Os, H1 and P1 fractions, and thuscomplementary treatments have to be carried out to separate and identifythem. Such treatments must be carried out on the biological test samplesto be tested before depositing them onto the gel.

[0013] Such treatments consist, for example, of thermal denaturing,incubation with specific inhibitors such as urea, amino acids, etc.,enzymatic incubation with neuraminidase, ficin, phospholipase C,incubation with specific antiplacental or anti-intestinal antisera.

[0014] Several separation procedures which are in current use have beendealt with by Van Hoof V. O., De Broe Marc, E., Clinical LaboratorySciences, vol. 31, issue 3 1994, “Interpretation and clinicalsignificance of alkaline phosphatase isoenzyme patterns”.

[0015] One particular procedure has been proposed in United Statespatent U.S. Pat. No. 5,264,098 which describes the separation of ALPisoenzymes using a gel electrophoresis reaction employing a gel buffercontaining at least one non ionic detergent and an anionic detergent.

[0016] Available treatments for identifying and quantifying ALPisoenzymes have certain disadvantages as regards routine analysis. Inaddition to high costs, they can on the one hand be long and canconsiderably complicate manipulation, and on the other hand, completedetermination (of all of the isoenzymes) necessitates a plurality oftreatments (2 or 3) for a single sample, limiting the number of sampleswhich can be simultaneously analysed on the one gel.

[0017] Other treatments have been proposed which, for example, recommendtreating the sample prior to loading onto the electrophoresis gel. Inthis regards, the action of the WGA lectin (wheat germ agglutinin) isparticularly interesting (see Sidney B. Rosalki, A. Ying Foo, ClinicalChemistry, 30/7, p. 1182-1186, 1984, “Two methods for separating andquantifying bone and liver alkaline phosphatase isoenzyme in plasma”,European patent EP-A-0 131 606 dated 5/11/86). EP-A-0 131 606 describesthe differential detection of bone and liver ALP isoenzyme comprisingtreating the test sample with lectin, then incubating the mixtureobtained followed by separating the ALP bound to the lectin from thefraction containing free ALP and determining the ALP activity in one ofthe two media or in both. In a particular implementation of that patent,the two fractions (ALP bound to lectin and free ALP) are separated byelectrophoresis.

[0018] With the exception of the intestinal forms, all isophosphatasespossess sialic acids and are thus affected by a treatment with WGAlectin to a greater or lesser extent. The osseous fraction is the mostsialated and thus is affected the most by this treatment, which undersuitable conditions retards its mobility and thus causes it toprecipitate in a zone which is distinct from the zone where the hepaticfraction is located.

[0019] In order to render ALP isoenzyme precipitation more selectivetowards the osseous isoenzyme, certain authors have used detergents suchas Triton X100 (Rosalki). However, despite the presence of suchdetergents, residual interactions of the WGA lectin with otherisophosphatases subsist, which cause co-precipitation of such fractionswith the osseous fraction.

[0020] In addition to this lack of specificity, a further disadvantageof this technique is to render the analysis considerably morecomplicated.

[0021] The publication by Rosalki S. B. et al, cited above,alternatively proposes incorporating lectin into the buffer used toimpregnate the electrophoresis gel prior to using this gel. Thisdispenses with prior treatment of the sample. In that case, the majorityof the osseous fraction is precipitated close to where the sample hasbeen loaded. The mobility of all of the other isoenzymes with theexception of intestinal isoenzymes is affected by the action of the WGAlectin despite the presence of the detergents mentioned above.

[0022] In the context of that treatment, the properties of the lectinused are its ability to interact specifically with the ALP isoenzymeswhich contain sialic acids.

SUMMARY OF THE INVENTION

[0023] The present invention proposes means for at least partiallyovercoming the disadvantages stated in prior art methods. In particular,the invention defines a method enabling separation and identification ofALP isoenzymes which is improved as regards specificity and sensitivity.

[0024] The present invention also provides consumers, in particularclinicians, with a process for separating, identifying and quantifyingthe principal alkaline isophosphatases, which process can be carried outin a single step on a single electrophoresis support which is easy toproduce.

[0025] The invention thus proposes a novel process for separating andidentifying ALP isoenzymes by electrophoresis, characterized in that thelectin is deposited on the electrophoresis support in a localisedmanner.

[0026] The lectin deposited in solution in a localised manner candiffuse into the support while remaining localised in a determined zoneof this support during electrophoretic migration.

[0027] The deposit in question, located close to the zone where thesample is deposited, is distinguished from the uniform loading over anextended zone or over the whole of the electrophoresis support asdescribed in the prior art.

[0028] The invention thus provides a process for separating alkalinephosphatase isoenzymes from a biological sample by electrophoresis,characterized in that the electrophoresis reaction is carried out on anelectrophoresis support after depositing a solution of lectin onto theelectrophoresis support in a given zone, under conditions which permitinteraction between said lectin and the ALP isoenzymes contained in theanalysed biological sample, deposition of the lectin solution furtherbeing carried out under conditions which are suitable to allowseparation of the ALP isoenzymes constituted by the osseous fraction andby the hepatic fraction.

[0029] The interaction in question leads to the formation of a complexbetween the lectin and the ALP isoenzyme until equilibrium is obtained.

[0030] The process of the invention enables the osseous fraction of theALP to be acted on in a manner which is more specific than on the otherALP fractions because of the reaction of this fraction with the lectin.

[0031] The biological sample analysed can be any biological sample whichmay contain ALP, in particular a biological fluid sample such as a serumor plasma sample, or possibly a tissue sample removed from a patient.

[0032] In the invention, electrophoresis is carried out on any suitableelectrophoresis support, in particular on a gel, more particularly on anagarose or a polyacrylamide gel, or on a porous membrane, in particularmade of cellulose acetate.

[0033] The particular conditions defined above for carrying out theelectrophoresis of the invention can be applied in the context of knownelectrophoresis methods which may or may not be automated.

[0034] The localised lectin deposit zone is determined as a function ofthe direction of migration of the sample and the lectin. Thus the lectindeposit zone is selected such that, during migration, the sampletraverses the lectin, the mobility of the latter during migration havingbeen taken into consideration. Similarly, the final position normallyreached by the other ALP isoenzymes is taken into consideration in orderto determine the lectin deposit zone with respect to that of the sample.In practice, the lectin and the sample are 1 to 10 mm apart,advantageously 5 mm, when the sample is loaded.

[0035] The other conditions for localised depositing of the lectin, suchas the concentration of the lectin, the time for application to theelectrophoresis support, are determined such that they enable osseousand hepatic ALP isoenzymes to be separated during electrophoresis underthe migration reaction conditions.

[0036] In other words, once the parameters of electrophoresis have beendetermined particularly as regards depositing the lectin, the process ofthe invention can separate the osseous and hepatic isoenzymes underconditions which are satisfactory for identifying them with respect tothe other ALP isoenzymes, and preferably to quantify them. The mobilityof the other ALP isoenzymes, affected during passage through the zonewhere the lectin is present, returns to normal outside this zone.

[0037] Since the osseous ALP fraction is more sialated, itselectrophoretic migration is the most affected by the passage of thesample through the lectin deposited on the support. As a result it isprecipitated in a zone which may be distinguished from the migrationzone of other ALP isoenzymes.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] In a preferred embodiment of the invention, a process forseparating alkaline phosphatase (ALP) isoenzymes by electrophoresis ischaracterized in that it comprises:

[0039] depositing the biological sample containing the ALP isoenzymes tobe separated on the electrophoresis support;

[0040] depositing a lectin solution which can interact with the ALPisoenzymes contained in the sample on the electrophoresis support;

[0041] applying an electric field to permit electrophoretic separationby migration of the ALP isoenzymes under conditions which can permitdifferential separation of the osseous and hepatic ALP fractions;

[0042] revealing the separated ALP isoenzymes.

[0043] Revealing is carried out using any known means, preferably usingan ALP substrate.

[0044] At the end of the steps resulting in separation of the ALPisoenzymes on the electrophoresis support, a quantitative analysis ofthe different separated isoenzymes, or certain of them, can be carriedout.

[0045] Different methods can be used to quantify the ALP isoenzymesdetected at the end of electrophoresis. As an example, the densitometryof the electrophoresis support can be measured after staining theseparated ALP fractions using an ALP substrate.

[0046] When the osseous ALP fraction is in excess with respect to thefixing capacity of the lectin deposited on the gel, the lectin may notprecipitate the whole of this fraction. In this case, the nonprecipitated portion continues to migrate with the other fractions, andcan be found in the form of a smear close to the subjacent isoenzymes,in particular H2, I1, I2 and I3. In this case, the different isoenzymescan be quantified by depositing the same sample a second time on thesame support, in the absence of lectin. On the “no lectin” pattern, thepercentages of the H1 Os P1 block and the separated fractions H2, I1,P2, I2, I3 and UF are determined. The percentage of the Os fraction isobtained by deducting the percentages of Hi and Pi determined from the“with lectin” profile from the H1 Os P1 block.

[0047] Within the context of the definitions given above, when carryingout electrophoresis in the presence of a localised deposit of lectin onthe electrophoresis support, the lectin and the sample can be depositedsimultaneously on the electrophoresis support.

[0048] In another embodiment of the invention, the sample and the lectinare deposited at different times.

[0049] Further, the sample and the lectin solution can respectively bedeposited on the electrophoresis support over periods which may beidentical or different.

[0050] Advantageously, within the context of the definitions givenabove, if necessary taken in combination, the lectin and the sample aredeposited over a substantially identical period of time, and preferablysimultaneously for practical and economic reasons.

[0051] For a given sample and a determined concentration of lectin inthe solution, the sample application and lectin application period, andthus the quantity of sample and lectin deposited on the electrophoresissupport, is selected so as to precipitate the Os fraction of the ALPisoenzymes such that the osseous and hepatic fractions are separatedduring migration. To determine the concentration of the lectin solutionand the period over which the solution is applied to the electrophoresissupport, the temperature reached by the support during migration is alsotaken into account.

[0052] Indeed, since the interaction of the lectin with the osseous ALPfraction is dependent on temperature, the temperature of theelectrophoresis support during the migration step must be taken intoaccount. A reduction in the lectin-osseous ALP interaction due to anincrease in the temperature of the electrophoresis support can becompensated for by an increase in the quantity of lectin deposited, forexample by increasing the concentration of the lectin solution used.

[0053] These parameters can be determined in the light of theindications below and the values given in the examples and can ifnecessary be adapted to the selected electrophoresis conditions, bycarrying out tests such as those which are given in the examples below.

[0054] The sample application and/or lectin solution application periodcan vary and can in particular be in the range of 5 to 20 min,preferably 15 minutes, for the sample and/or for the lectin.

[0055] Depositing can be carried out using any known manual or automatedmeans, for example using “comb” type applicators, for example thedevices described in European patent application EP-A-0 493 996.

[0056] The lectin used is in the form of an aqueous solution.

[0057] Thus the concentration of lectin deposited on the electrophoresissupport used under normal conditions is in the range of 0.1 mg/ml to thelimit of solubility of lectin in water. This concentration isadvantageously in the range of 0.5 to 15 mg/ml, preferably in the rangeof 1 to 10 mg/ml.

[0058] In a particular implementation of the invention, the process ischaracterized in that when the lectin concentration is in the range of 1to 10 mg/ml, the migration temperature is respectively in the range of18° C. to 53° C. These conditions are applicable inter alia todepositing a lectin solution over a period of close to about 15 minuteswith a comb type applicator as described in European patent applicationEP-A-0 493 996.

[0059] The invention particularly concerns an implementation of anelectrophoresis process in which, when the ALP isoenzymes present in thetest sample have an electrophoretic mobility in the direction of theanode, the lectin is deposited between the anode and the sample depositzone.

[0060] Advantageously in this case, the lectin is deposited between thebiological sample deposit point and the zone normally occupied byintestinal fraction 3 (I3) at the end of the migration step, when thisfraction is present in the sample.

[0061] Different lectins can be used in the invention. Lectins areproteins which fix sialated groups. Lectins which can be used in thepresent invention which can be cited include wheat germ (Triticumvulgaris) lectin, or WGA (Wheat Germ Agglutinin). WGA lectin can beobtained from Sigma, Pharmacia, etc..

[0062] The invention also concerns a process satisfying the definitionsgiven above, taken separately or in combination, in which separation offractions other than the osseous and hepatic 1 of ALP is improved.

[0063] The invention thus makes available, under conditions which arecompatible with routine laboratory analyses, means for in vitrodetection in a biological sample of the abnormal presence of one or moreALP isoenzymes.

[0064] This process can in particular be carried out during a protocolfor diagnosing a hepatic or biliary disorder corresponding to theabnormal presence of the hepatic ALP fraction. The invention can alsoenable the abnormal presence of the osseous fraction to be researched inthe context of the diagnosis of osseous disorders.

[0065] The invention also concerns kits for carrying out separation offractions constituted by ALP isoenzymes by electrophoresis.

[0066] The process of the invention thus has the advantage of using asingle electrophoresis support, without prior treatment of the sample,to carry out qualitative determination of all of the ALP isoenzymes inone deposit and their quantitative determination in two deposits onto asingle support. The electrophoresis support is free of lectin prior toits use, which enables it to be stored under the usual temperatureconditions and does not change its manufacturing cost.

[0067] A kit comprises, for example:

[0068] an electrophoresis support comprising a porous material suitablefor depositing a biological sample to be analysed and for carrying outelectrophoretic migration;

[0069] a solution of lectin in a concentration in the range of 0.1 mg/mlto 15 mg/ml, advantageously in the range of 1 to 10 mg/ml, preferably inthe range of 1 to 10 mg/ml.

[0070] The kit of the invention can also comprise the buffer or buffersrequired for the electrophoresis reaction. It can also contain ALPactivity revealing reagents.

[0071] Further advantages and features of the invention will become moreclear from the following examples.

EXAMPLES

[0072] Principles of the reaction using lectin when it is uniformlydistributed in the electrophoresis gel

[0073] The interaction of the WGA lectin with a slightly sialatedisophosphatase can be represented by the equilibrium:

[0074] (A) Isophosphatase+WGA

{isophosphatase-WGA} complex

[0075] Under normal electrophoresis conditions, namely at a basic pH,isophosphatases have an anodic mobility while lectin has a slightlycathodic mobility. The anodic mobility of the {isophosphatase-WGA}complex will thus be lower than that of free isophosphatase.

[0076] During the whole of the migration period, the isophosphatase willinteract with the WGA lectin in accordance with the equilibrium ofreaction (A) and thus will be slowed down.

[0077] Thus the pattern obtained on the gel with no WGA lectin where,under the given conditions, all fractions are separated with theexception of the block constituted by the H1, Os (possibly P1)isoenzymes, will lead, for a gel incorporating WGA lectin over itsentire surface, to a pattern where the H1 (possibly P1) will bedisengaged from the osseous but where the H1, P1 fractions on the onehand and the H2, I2 fractions on the other hand, which are resolved onthe gel without WGA lectin, will be merged. It would then be necessary,even for a simple qualitative estimation of the isoenzymes of a sample,to carry out electrophoresis on two types of gel, which would thereforecomplicate the analysis.

[0078] In addition to this disadvantage, WGA lectin is heat sensitivewhich renders the production of gels containing it very difficult;further, consumers are obliged to store these gels between 4° C. and 8°C. to preserve their performance intact.

[0079] Principles of the reaction of the invention, using lectindeposited onto the electrophoresis gel in a localised manner

[0080] In the invention, a solution of WGA lectin is deposited in frontof the sample, i.e., between the sample and the anode. Theelectrophoresis gel is thus not impregnated with lectin over its entiresurface. The WGA lectin may or may not be deposited simultaneously withthe sample. Once both deposits have been carried out, the voltage isapplied to obtain electrophoretic separation.

[0081] Under these conditions, the major portion of the Os fractionprecipitates out when it goes through the zone where the WGA lectin hasbeen deposited. The electrophoretic mobility of the other isophosphatasefractions is affected by the WGA lectin in accordance with theequilibrium of the reaction: Isophosphatase+WGA

{isophosphatase-WGA} complex, but only when traversing the zonecontaining the WGA lectin. This zone is very reduced in size (<1 mm)which means that the profile remains practically identical to thatobtained in the absence of lectin, with the exception of the osseousfraction which is disengaged from the H1 and P1 fractions. Under theseconditions, electrophoresis of the sample with the deposited lectin infront of the sample permits qualitative analysis on a single gel in asingle step: all of the isoenzymes can be identified from their positionand without the need for complementary treatments to be carried out onthe sample.

[0082] In the case when the different isoenzymes which have beenseparated and revealed by a suitable reagent have to be quantified,account must be taken of the fact that precipitation of the osseousfraction by the WGA lectin is not complete, in particular in the casewhere this osseous fraction is highly increased. A portion of theosseous alkaline phosphatase molecules escape precipitation when goingthrough the lectin deposit. The Os-WGA bond is, however, sufficient forthese non-precipitated osseous alkaline phosphatase molecules to entrainWGA lectin with them. They are thus sufficiently slowed so that they donot reach the H1P1 zone. Thus in addition to the precipitation curve ofthe osseous fraction, a smear of osseous alkaline phosphatase isobtained which reaches to the zone to which the H2 migrates. Under theseconditions, quantification of the isoenzymes subjacent to this streak,namely the isoenzymes H2, I1, I2, I3, is disturbed by this smear.

[0083] To enable quantification in a situation where the quantity of theOs fraction risks being higher than that which can precipitate, twodeposits of the same sample should be made side by side, one with thelectin deposit, the other without. The H1 and P1 fractions can bequantified on the pattern with the WGA lectin. The percentages of the H1Os P1 block and the separated H2, I1, P2, I2, I3 and UF fractions aredetermined using the pattern with no lectin. The percentage of the Osfraction is obtained by subtracting the H1 and P1 percentages determinedusing the profile with lectin from the H1 Os P1 block.

[0084] Thus a single gel can be used to determine, in a single step withtwo loads, the percentage of all of the isoenzymes with no priortreatment of the sample.

Concentration of lectin and temperature of electrophoresis.

[0085] The concentration of lectin to be used and the migrationtemperature (temperature of the electrophoresis support) are closelylinked. The equilibrium for formation of the isophosphatase-WGA lectincomplex is indeed temperature-dependent. Thus increasing the temperatureencourages dissociation of the complex. The lectin concentration must beincreased to obtain the same precipitating power of the osseousfraction.

[0086] In practice, the concentration of the lectin to be used is afunction of the gel and can be selected using the following table:Temperature of gel during migration Concentration of lectin, ° C. mg/ml18  1 28  2 38  3 48  5 53 10

[0087] Thus if the process is carried out in accordance with theinvention with an electrophoresis system which cannot control themigration temperature (Example 1), the lectin concentration has to beincreased to take account of the maximum temperature achieved in the gelduring migration. This temperature depends on a number of parameterssuch as the ionic strength and the dimensions of the gel, the migrationparameters and the external temperature during migration. Under theconditions used in Example 1, the maximum temperature reached by the gelis close to 38° C., and thus a lectin concentration of 3 mg/ml is used.In Example 2, the instrument can regulate the electrophoresistemperature at 20° C. However, when the electrophoresis is carried outat a constant power of 20 W, the effective temperature of the gel is 28°C. As a result, the concentration of lectin necessary to obtain thedesired effect is 2 mg/ml.

Position and duration of lectin depositing

[0088] Under the pH conditions (basic) used for electrophoresis, themobility of all of the alkaline isophosphatases is in the anodedirection; in contrast, lectin has a very slight mobility in the cathodedirection. In order for the osseous fraction to encounter the lectin,the lectin has to be deposited between the sample and the anode. Moreprecisely, it must be deposited at a distance which is less than orequal to that traversed by the osseous fraction under the migrationconditions used and in the absence of lectin. However, in order toprevent precipitation of the osseous fraction in a zone already occupiedby other fractions, it appears more judicious to deposit the lectin at adistance between the sample and the position which the I3 fractionoccupies at the end of migration. In practice, the lectin is depositedin front of the sample at a distance in the range of 1 to 10 mm,preferably close to 5 mm.

[0089] The lectin is deposited at the same time or after the sample. Thetwo do not have to be deposited simultaneously. However, if the sampleand the lectin are not deposited at the same time, care must be taken toprevent diffusion of the first deposit when depositing the second. Forthis reason it is easier to deposit the sample and the lectinsimultaneously and over the same period. Further, the two deposits areparallel to each other and perpendicular to the direction of migration.

EXAMPLE N° 1

[0090] Gel with the following composition: Agarose  1% Tris  0.03 MSodium Barbital  0.025 M Barbital acid  0.005 M Sodium azide  1 g/lTriton X 100 10 g/l Nonidet NP 40  5 g/l

[0091] 40 ml of demineralised water and 0.5 g of agarose were introducedinto a 100 ml Erlenmeyer flask. After boiling for 5 minutes withconstant stirring, the agarose had dissolved to produce a perfectlyclear solution. The temperature of this solution was reduced to 50° C.in a thermostated bath. 10 ml of a concentrated buffer solutioncontaining 18 g/l of Tris, 27.75 g/l of sodium Barbital, 4.6 g/l ofBarbital acid, 5 g/l of sodium azide, 50 g/l of Triton X 100 and 25 g/lof Nonidet NP 40 were introduced into a 50 ml Erlenmeyer flask. Thissolution was maintained at 50° C. in the thermostatted bath.

[0092] The pre-heated buffer was added to the agarose solution. It washomogenised and maintained at 50° C.

[0093] 5 ml of the above solution, removed with a pipette, was thenuniformly poured onto a 10×8 cm hydrophilic plastic sheet.

[0094] After gelling and stabilisation, the gel could be used. Freshsera to be analysed were deposited in 2 adjacent deposits, 2.5 cm fromthe edge, on the cathode side. A 3 mg/ml solution of Wheat GermAgglutinin (WGA) lectin was applied simultaneously and over the sameperiod 32 mm in front of one of the 2 deposits of each sample, i.e.,between the sample and the anode. The application time could be 15minutes for each deposit made with a microporous membrane applicator assold by Sebia, described in European patent application EP-A-0 493 996.

[0095] The alkaline isophosphatases were separated by electrophoresis ina vessel the tanks of which contained a Tris 0.003 M, sodium Barbital0.025 M, Barbital acid 0.005 M, sodium azide 0.1 g/l buffer for a periodof 50 minutes at a constant voltage of 100 V.

[0096] Incubating the gel with a conventional substrate for this enzyme(for example bromochloroindolyl phosphate and nitrobluetetrazolium) thenrevealed the alkaline phosphatase activities. A blue stain was thusobtained at the location of each isophosphatase fraction, proportionalto its activity. After revealing, the gel was washed then dried andanalysed by densitometry to quantify the different alkalineisophosphatases.

EXAMPLE N° 2

[0097] Gel with the following composition: Agarose  1% Tris  0.38 MBoric acid  0.06 M sodium azide  1 g/l Triton X 100 10 g/l Nonidet NP 40 5 g/l

[0098] 40 ml of demineralised water and 0.5 g of agarose were introducedinto a 100 ml Erlenmeyer flask. After boiling for 5 minutes withconstant stirring, the agarose had dissolved to produce a perfectlyclear solution. The temperature of this solution was reduced to 50° C.in a thermostatted bath. 10 ml of a concentrated buffer solutioncontaining 229.9 g/l of Tris, 18.55 g/l of boric acid, 5 g/l of sodiumazide, 50 g/l of Triton X 100 and 25 g/l of Nonidet NP 40 wereintroduced into a 50 ml Erlenmeyer flask. This solution was maintainedat 50° C. in the thermostatted bath.

[0099] The pre-heated buffer was added to the agarose solution. It washomogenised and maintained at 50° C. 5 ml of the above solution, removedwith a pipette, was then uniformly poured onto a 10×8 cm hydrophilicplastic sheet.

[0100] After gelling and stabilisation, the gel could be used. Freshsera to be analysed were deposited in 2 adjacent deposits, 2.5 cm fromthe edge, on the cathode side. A 2 mg/ml solution of Wheat GermAgglutinin (WGA) lectin was applied simultaneously and over the sameperiod 5 mm in front of one of the 2 deposits of each sample, i.e.,between the sample and the anode.

[0101] The alkaline isophosphatases were separated by electrophoresis inan apparatus which could adjust the temperature to 20° C. Migration wascarried out at a constant power of 20 W for 20 minutes.

[0102] The alkaline phosphatase activities and densitometry weremeasured as in the previous example.

1. A process for separating alkaline phosphatase (ALP) isoenzymes from abiological sample by electrophoresis, wherein the electrophoresis iscarried out on an electrophoresis support after depositing a solution oflectin onto the electrophoresis support in a given localised zone, underconditions which permit interaction between said lectin and the ALPisoenzymes contained in the analysed biological sample, deposition ofthe lectin solution further being carried out under conditions which aresuitable to allow separation of the ALP isoenzymes constituted by theosseous fraction and by the hepatic fraction.
 2. A process forseparating alkaline phosphatase (ALP) isoenzymes by electrophoresis saidprocess comprising the steps of: depositing the biological samplecontaining the ALP isoenzymes to be separated on the electrophoresissupport; depositing a lectin solution which can interact with the ALPisoenzymes contained in the sample on the electrophoresis support;applying an electric field to permit electrophoretic separation bymigration of the ALP isoenzymes under conditions which can permitdifferential separation of the osseous and hepatic ALP fractions; andrevealing the separated ALP isoenzymes.
 3. The process for separation byelectrophoresis according to claim 1 or claim 2 , wherein theelectrophoretic separation of the ALP isoenzymes is followed by a stepof quantitative analysis of the different separated ALP isoenzymes. 4.The process for separating alkaline phosphatase (ALP) isoenzymes byelectrophoresis according to claim 1 or claim 2 , wherein the period fordepositing the lectin solution and/or the period for depositing thesample is/are in the range of 5 to 20 minutes.
 5. The process forseparating alkaline phosphatase (ALP) isoenzymes by electrophoresisaccording to claim 1 or claim 2 , wherein the concentration of thelectin solution is determined as a function of the temperature of theelectrophoresis support and the period over which the lectin solution isapplied to the support.
 6. The process for separating alkalinephosphatase (ALP) isoenzymes by electrophoresis according to claim 1 orclaim 2 , wherein the concentration of the lectin solution is in therange of 0.1 mg/ml to the limit of solubility in water.
 7. The processfor separating alkaline phosphatase (ALP) isoenzymes by electrophoresisaccording to claim 1 or claim 2 , wherein the concentration of thelectin solution is in the range of 1 to 10 mg/ml.
 8. The process forseparating alkaline phosphatase (ALP) isoenzymes by electrophoresisaccording to claim 1 or claim 2 , wherein the migration temperature isin the range of 18° C. to 53° C. respectively.
 9. The process forseparating alkaline phosphatase (ALP) isoenzymes by electrophoresisaccording to claim 1 or claim 2 , wherein the concentration of thelectin solution is determined as a function of the maximum temperatureachieved in the electrophoresis support during the migration step. 10.The process for separating alkaline phosphatase (ALP) isoenzymes byelectrophoresis according to claim 1 or claim 2 , wherein the mobilityof the ALP isoenzymes is in the anode direction and the lectin isdeposited between the anode and the biological sample.
 11. The processfor separating alkaline phosphatase (ALP) isoenzymes by electrophoresisaccording to claim 2 , wherein the lectin is deposited between thebiological sample and a zone normally occupied by an intestinal fraction3 (I3) at the end of the migration step.
 12. The process for separatingalkaline phosphatase (ALP) isoenzymes by electrophoresis according toclaim 1 or claim 2 , wherein the lectin is wheat germ lectin (wheat germagglutinin or WGA).
 13. The process for separating alkaline phosphatase(ALP) isoenzymes by electrophoresis according to claim 1 or claim 2 ,wherein the electrophoresis support is an agarose gel or polyacrylamide.14. The process for separating alkaline phosphatase (ALP) isoenzymes byelectrophoresis according to claim 1 or claim 2 , wherein theelectrophoresis support is a cellulose acetate membrane.
 15. The processfor separating alkaline phosphatase (ALP) isoenzymes by electrophoresisaccording to claim 3 , wherein said quantitative analysis is carried outby measuring the densitometry of the electrophoresis support afterstaining the ALP fractions using an ALP substrate.
 16. A process fordetecting the abnormal presence of one or more alkaline phosphatase(ALP) isoenzymes in a biological sample by electrophoresis, wherein theelectrophoresis is carried out on an electrophoresis support afterdepositing a solution of lectin onto the electrophoresis support in agiven localised zone, under conditions which permit interaction betweensaid lectin and the ALP isoenzymes contained in the analysed biologicalsample, deposition of the lectin solution further being carried outunder conditions which are suitable to allow separation of the ALPisoenzymes constituted by the osseous fraction and by the hepaticfraction.
 17. A process for detecting the abnormal presence of one ormore alkaline phosphatase (ALP) isoenzymes in a biological sample byelectrophoresis, said process comprising the steps of: depositing thebiological sample containing the ALP isoenzymes to be separated on theelectrophoresis support; depositing a lectin solution which can interactwith the ALP isoenzymes contained in the sample on the electrophoresissupport; applying an electric field to permit electrophoretic separationby migration of the ALP isoenzymes under conditions which can permitdifferential separation of the osseous and hepatic ALP fractions; andrevealing the separated ALP isoenzymes.
 18. The process according toclaim 16 or claim 17 , wherein said abnormal presence of one or morealkaline phosphatase (ALP) isoenzymes is indicative of a hepatic or abilary disorder.
 19. The process according to claim 16 or claim 17 ,wherein said abnormal presence of one or more alkaline phosphataseisoenzymes is indicative of an osseous disorder.
 20. A kit for carryingout separation by electrophoresis, which comprises: an electrophoresissupport comprising a porous material suitable for depositing abiological sample to be analyzed and for carrying out electrophoreticmigration; and a solution of lectin.
 21. The kit according to claim 20 ,wherein said solution of lectin has a concentration in the range of 0.1mg/ml to 15 mg/ml.